BABAM2 Antibody

What is BABAM2 and why is it significant in research?

BABAM2 is an anti-apoptotic, death receptor-associated protein that interacts with tumor necrosis factor-receptor-1. It functions as an adapter protein in multiple complexes, including the BRCA1-A complex and BRISC complex. The significance of BABAM2 lies in its role within the BRCA1-A complex, which specifically recognizes 'Lys-63'-linked ubiquitinated histones H2A and H2AX at DNA lesion sites, directing the BRCA1-BARD1 heterodimer to double-strand breaks (DSBs) . Additionally, BABAM2 plays a crucial role in regulating cell cycle progression and maintaining pluripotency status following DNA damage, making it an important target in cancer and stem cell research .

What are the common synonyms and alternative names for BABAM2?

When searching literature or ordering antibodies, researchers should be aware that BABAM2 is also known by several other names, including:

• BRCC45 (BRCA1/BRCA2-containing complex subunit 45)

• BRE (BRCA1-A complex subunit BRE)

• BRCC4
  These alternative designations reflect the protein's functional associations with different complexes and pathways .

What is the molecular structure and localization of BABAM2 protein?

The canonical human BABAM2 protein consists of 383 amino acid residues with a molecular mass of approximately 43.6 kDa. Up to four different isoforms have been reported for this protein. Regarding subcellular localization, BABAM2 is found in both the nucleus and cytoplasm, consistent with its diverse functions in DNA damage response and anti-apoptotic signaling . Immunofluorescence staining can be used to visualize its localization pattern in different cell types and under various experimental conditions .

Which applications are BABAM2 antibodies commonly used for?

BABAM2 antibodies are employed in multiple experimental applications, including:

• Western Blot (most widely used)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

These applications enable researchers to detect, quantify, and visualize BABAM2 expression in various experimental settings, from protein lysates to fixed cells and tissue sections .

How are BABAM2 antibodies used in cancer research?

BABAM2 antibodies serve as valuable tools in cancer research, particularly for the immunohistochemical detection of breast cancer, cervical cancer, and rectal cancer . The mRNA and protein expression of BABAM2 can be detected in a variety of cancer types. Recent studies have identified BABAM2-ALK fusion in gynecological clear cell carcinoma through next-generation sequencing (NGS), suggesting a potential pathogenic mechanism in this aggressive tumor type that typically shows high resistance to conventional platinum-based chemotherapy . Furthermore, BABAM2 expression has been associated with favorable prognosis in breast cancer patients receiving radiation therapy, highlighting its potential value as a prognostic biomarker .

What is the role of BABAM2 in DNA damage response pathways?

BABAM2 functions as a component of the BRCA1-A complex, which possesses deubiquitinase activity that specifically removes 'Lys-63'-linked ubiquitin on histones H2A and H2AX at DNA damage sites . In conjunction with RAD51, BRCA1, and BRCA2, BABAM2 forms the BRCA1-BRCA2-containing complex (BRCC), a holoenzyme complex with E3 ubiquitin ligase activity that repairs double-strand breaks in DNA . Through binding to Rap80, this complex is recruited to DNA damage sites. BABAM2's involvement in these complexes makes it a critical player in maintaining genomic stability, with implications for cancer development and therapeutic responses .

How does BABAM2 influence cell cycle regulation and pluripotency?

Research using BABAM2 knockout models has demonstrated that BABAM2 plays an essential role in promoting cell cycle progression and preventing cellular senescence. In mouse embryonic stem cells (mESCs), BABAM2 deficiency leads to decreased expression of CDK2 and CDC25A following DNA damage, inhibiting G1 phase progression . The expression patterns of these cell cycle regulators correlate with BABAM2 status, indicating that BABAM2 might stabilize CDC25A upon DNA damage, thereby promoting G1 phase cell cycle progression through CDK2 upregulation . Additionally, BABAM2 absence affects p53 response to gamma irradiation, with prolonged and elevated p53 expression observed in BABAM2-deficient cells, which subsequently inhibits NANOG expression and impacts pluripotency maintenance .

What are the critical considerations when selecting a BABAM2 antibody for research?

When selecting a BABAM2 antibody, researchers should consider:

• Antibody Specificity: Verify that the antibody specifically recognizes BABAM2 without cross-reactivity to related proteins.

• Application Compatibility: Ensure the antibody is validated for your intended application (WB, IF, IHC, etc.).

• Species Reactivity: Confirm the antibody recognizes BABAM2 in your experimental species.

• Epitope Recognition: Consider which domain or region of BABAM2 the antibody targets, especially if studying specific isoforms.

• Validation Data: Review the manufacturer's validation data and literature citations.

• Clonality: Choose between monoclonal (higher specificity) or polyclonal (potentially better signal) based on experimental needs.

Proper antibody selection is crucial for obtaining reliable and reproducible results in BABAM2-related research .

What protocols are recommended for detecting BABAM2 in Western blot experiments?

For optimal Western blot detection of BABAM2, the following protocol is recommended based on published research:

• Sample Preparation:

  • Harvest cells in ice-cold cell lysis buffer containing 1 mM PMSF, 1 mM DTT, and 1× protease inhibitor

  • Isolate supernatant by centrifugation at 13,000 rpm for 10 min at 4°C

  • Determine protein concentration using a BCA Protein Assay Kit

• Gel Electrophoresis and Transfer:

  • Load 20 μg of protein per lane on SDS-PAGE gels

  • Transfer proteins onto nitrocellulose membranes

• Antibody Incubation:

  • Block membranes with 5% non-fat milk or BSA for 1 hour at room temperature

  • Incubate with anti-BABAM2 primary antibody (1:1000 dilution) overnight at 4°C

  • Wash membranes and incubate with appropriate secondary antibody

• Detection:

  • Develop using enhanced chemiluminescence

  • Analyze band intensity at approximately 43.6 kDa, corresponding to BABAM2

This protocol has been successfully used in studies examining BABAM2's role in DNA damage response and cell cycle regulation .

What are the recommended procedures for immunofluorescence staining of BABAM2?

For immunofluorescence detection of BABAM2 in cultured cells, researchers should follow this optimized protocol:

• Cell Preparation:

  • Seed cells onto appropriate coverslips (e.g., gelatin-coated for stem cells)

  • Allow cells to grow to desired confluence

• Fixation and Permeabilization:

  • Wash cells with PBS

  • Fix in 4% paraformaldehyde for 15 min at room temperature

  • Permeabilize using 0.1% Triton X-100 in PBS for 30 min

• Blocking and Antibody Incubation:

  • Block with 10% horse serum in PBST (0.1% Tween-20 in PBS) for 1 hour

  • Incubate with anti-BABAM2 primary antibody (1:200 dilution) overnight at 4°C

  • Wash three times with PBST

  • Incubate with appropriate Alexa Fluor-conjugated secondary antibody (1:5000) for 1 hour at room temperature in the dark

• Nuclear Counterstaining and Imaging:

  • Counterstain nuclei with 5 μg/mL DAPI

  • Mount slides and image using confocal microscopy

This protocol allows visualization of BABAM2's subcellular localization and enables co-localization studies with other proteins of interest .

How can researchers investigate BABAM2's role in DNA damage response using antibody-based approaches?

To investigate BABAM2's role in DNA damage response, researchers can employ several antibody-based approaches:

• Co-immunoprecipitation (Co-IP): Use BABAM2 antibodies to pull down protein complexes and identify interaction partners before and after DNA damage induction. This approach can reveal dynamic changes in BABAM2-containing complexes such as BRCA1-A and BRISC.

• Chromatin Immunoprecipitation (ChIP): Apply BABAM2 antibodies in ChIP assays to determine if BABAM2 is recruited to specific DNA damage sites, potentially in conjunction with BRCA1/BRCA2.

• Proximity Ligation Assay (PLA): Utilize BABAM2 antibodies alongside antibodies against suspected interaction partners to visualize protein-protein interactions in situ following DNA damage.

• Immunofluorescence after DNA Damage: Compare BABAM2 localization before and after treating cells with DNA-damaging agents such as gamma irradiation (8 Gy) or doxorubicin (250 nM for 6 hours) .

These methodologies can provide insights into how BABAM2 contributes to DNA damage sensing, signaling, and repair mechanisms.

What experimental approaches can demonstrate the functional relationship between BABAM2 and cell cycle regulation?

To establish the functional relationship between BABAM2 and cell cycle regulation, researchers can implement these experimental approaches:

• BABAM2 Knockout/Knockdown Studies: Generate BABAM2-deficient cell lines using CRISPR-Cas9 or siRNA technology, then analyze cell cycle distribution using flow cytometry.

• Cell Proliferation Assays: Compare proliferation rates between wild-type and BABAM2-deficient cells using assays such as CCK-8, which has been previously used to demonstrate growth defects in BABAM2-knockout cells .

• Cell Cycle Marker Analysis: Examine the expression and localization of cell cycle regulators (e.g., CDK2, CDC25A) in response to BABAM2 manipulation using Western blot and immunofluorescence .

• DNA Damage Recovery Experiments: Assess how BABAM2 affects cell cycle recovery after DNA damage by monitoring checkpoint activation and resolution in wild-type versus BABAM2-deficient cells.

• Rescue Experiments: Reintroduce wild-type or mutant BABAM2 into knockout cells to identify domains critical for cell cycle regulation.

These approaches have revealed that BABAM2 promotes G1 phase cell cycle progression by upregulating CDK2 through stabilizing CDC25A after DNA damage induction .

How can researchers investigate the interaction between BABAM2 and other BRCA1-associated proteins?

To study interactions between BABAM2 and other BRCA1-associated proteins, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use BABAM2 antibodies to pull down protein complexes, then probe for BRCA1, BRCA2, RAD51, and other components of the BRCC complex.

• Mass Spectrometry Following IP: Perform mass spectrometry analysis on proteins co-precipitated with BABAM2 to identify novel interaction partners.

• GST Pull-down Assays: Express BABAM2 as a GST-fusion protein to identify direct binding partners among BRCA1-associated proteins.

• Yeast Two-Hybrid Screening: Map specific interaction domains between BABAM2 and other complex components.

• FRET or BiFC Analysis: Visualize protein-protein interactions in living cells by tagging BABAM2 and potential partners with appropriate fluorescent proteins.

These techniques can elucidate how BABAM2 contributes to the formation and function of the BRCC complex, which is known to repair double-strand breaks in DNA .

What are common technical challenges when working with BABAM2 antibodies and how can they be addressed?

Researchers working with BABAM2 antibodies may encounter several technical challenges:

Addressing these challenges requires careful optimization and validation of antibodies for specific experimental contexts .

How can researchers validate the specificity of BABAM2 antibodies?

To ensure BABAM2 antibody specificity, researchers should implement these validation strategies:

• Positive and Negative Controls:

  • Use cell lines with known BABAM2 expression levels as positive controls

  • Include BABAM2 knockout or knockdown samples as negative controls

• Peptide Competition Assay:

  • Pre-incubate the antibody with a blocking peptide containing the target epitope

  • If specific, the signal should be significantly reduced or eliminated

• Multiple Antibody Comparison:

  • Test multiple antibodies targeting different BABAM2 epitopes

  • Consistent results across antibodies suggest specificity

• Molecular Weight Verification:

  • Confirm that the detected band appears at the expected molecular weight (43.6 kDa for canonical BABAM2)

  • Be aware of potential isoforms (up to 4 have been reported)

• Recombinant Protein Controls:

  • Test antibody against purified recombinant BABAM2

  • Compare with detection in complex biological samples

These validation steps are critical for generating reliable and reproducible research findings.

What new insights have emerged regarding BABAM2's role in cancer biology?

Recent studies have revealed several important advances in understanding BABAM2's role in cancer:

• BABAM2-ALK Fusion: Next-generation sequencing has identified BABAM2-ALK fusion in gynecological clear cell carcinoma, suggesting a novel pathogenic mechanism in this aggressive tumor type that typically shows high resistance to conventional platinum-based chemotherapy .

• Prognostic Value: BABAM2 expression has been associated with favorable prognosis in breast cancer patients receiving radiation therapy, indicating its potential utility as a biomarker for treatment response prediction .

• Anti-Apoptotic Mechanism: Research has demonstrated that BABAM2 can bind to Fas and TNF-R1 to prevent downstream signaling and inhibit activation of the mitochondrial apoptotic pathway. Additionally, BABAM2 can exert anti-apoptotic effects by regulating XIAP expression .

• Interaction with PPDPF: Mechanistic studies have shown that PPDPF (Pancreatic Progenitor Cell Differentiation and Proliferation Factor) interacts with BABAM2 and blocks its ubiquitination by MDM2, thereby stabilizing BABAM2 and promoting cancer progression .

These findings highlight BABAM2's multifaceted roles in cancer development and treatment response, opening new avenues for diagnostic and therapeutic development.

What emerging applications of BABAM2 antibodies are being developed for translational research?

Emerging applications of BABAM2 antibodies in translational research include:

• Biomarker Development: BABAM2 antibodies are being evaluated for their utility in detecting BABAM2 expression as a prognostic or predictive biomarker in multiple cancer types, particularly breast cancer, cervical cancer, and rectal cancer .

• Therapeutic Response Prediction: Given BABAM2's role in DNA damage response, antibody-based detection methods are being developed to predict patient responses to radiation therapy and DNA-damaging chemotherapeutics .

• Companion Diagnostics: As targeted therapies against DNA repair pathways emerge, BABAM2 antibody-based assays could serve as companion diagnostics to identify patients likely to benefit from specific treatments.

• Circulating Tumor Cell Detection: Advanced applications may include using BABAM2 antibodies to detect and characterize circulating tumor cells in liquid biopsies.

These translational applications build upon fundamental research findings and may ultimately contribute to more personalized approaches to cancer diagnosis and treatment.